CN110055263A - Encode gene C ry1Ab-MR, its expression vector and its application of Bt insecticidal proteins - Google Patents
Encode gene C ry1Ab-MR, its expression vector and its application of Bt insecticidal proteins Download PDFInfo
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- CN110055263A CN110055263A CN201910190603.5A CN201910190603A CN110055263A CN 110055263 A CN110055263 A CN 110055263A CN 201910190603 A CN201910190603 A CN 201910190603A CN 110055263 A CN110055263 A CN 110055263A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The invention discloses the genes of a kind coding Bt insecticidal proteinsCry1Ab‑MR, its expression vector and its application, it is intended to solve anti insect gene low technical problem of expression silencing, transgenic corns insect resistace in corn.The present invention devises a kind of anti insect geneCry1Ab‑MRAnd its cloning primer；It further devises and a kind of contains anti insect gene for genetic transformationCry1Ab‑MRProtokaryon and carrier for expression of eukaryon；The protokaryon and eukaryotic expression engineering bacteria for genetic transformation have been constructed out simultaneously.And the application by anti insect gene, carrier for expression of eukaryon, engineering bacteria in transgenic plants.The anti insect gene that the present invention provides for the first timeCry1Ab‑MRIt can be expressed in maize seed, and the insect resistace of maize leaf can be significantly provided, and can avoid the generation of gene silencing phenomenon, there are wide application space and market prospects providing plant resistance to insect.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of gene for encoding Bt insecticidal proteinsCry1Ab-MR、
Its expression vector and its application.
Background technique
Bacillus thuringiensis (Bacillus thuringiensis, Bt) and belong to prokaryotes bacterium guiding principle Bacillaceae
Bacillus is a kind of gram-positive bacteria.It is separated from the silkworm body fluid caught an illness within 1901, finds it to part
Lepidopterous insects have toxic action.Its desinsection principle is: during forming gemma, producing a series of pairs of insects has
The insecticidal protein crystal of high degree of specificity toxic action, since insecticidal protein crystal itself does not have bioactivity, referred to as former poison
Element, after sensibility insect absorbs parent toxin, under middle intestines alkaline condition, parent toxin is hydrolyzed to polypeptide, these polypeptides have spy
Different insecticidal activity can be irreversibly inserted into plasma membrane, make in conjunction with the specific receptor of the columnar cell of middle intestines gut epithelium
Duct is formed on cell membrane, and then destroys the ionic equilibrium of cell, is eventually led to cell cracking, is caused insect death.Yin Qibiao
Up to product Bt toxalbumin good disinsection effect, it is safe and efficient the advantages that and become be most widely used turn killing gene.
As the technologies such as the development of Protocols in Molecular Biology and gene cloning, DNA operation occur, the mankind start from Bt bacterium
Its toxoprotein gene is cloned into engineering bacteria, genetically modified organism is grown rapidly.From over 1987, the anti insect gene of Bt is
In successful conversion various plants, resistant transgenic plant is obtained, wherein much having entered field experiment, or even some has been opened
The plantation of beginning large area.
But the insect resistace of the genetically modified plants obtained previously is very low, the content of toxalbumin is also very low, is not able to satisfy production
On actual demand, be difficult to apply mostly；With the widespread adoption of anti insect gene, insect generates resistance to insecticidal proteins；It is anti-
The pest-resistant spectrum of worm gene is narrow；There are gene " silencing " phenomenons, etc. in the intracorporal expression of plant for foreign gene.It is above-mentioned to turn Bt poison egg
The potential problems of the zoophobous of white gene in the application increasingly appear, and influence its sustainable utilization.
With deepening continuously for engineering of insect-resistant plant, at present in gene modification and transformation, the building of expression vector, plant
Cultivation of transform mode, zoophobous of object tissue etc. becomes focus on research direction and subtracts to cultivate anti-pest crop
Few environmental pollution, protects human health.
Summary of the invention
The technical problem to be solved in the present invention is to provide the genes of a kind coding Bt insecticidal proteinsCry1Ab-MR, its table
Up to carrier and its application, to solve anti insect gene low technical problem of expression silencing, transgenic corns insect resistace easily in corn.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
Based on long-term a large number of experiments and practical experience, a kind of anti insect gene is designedCry1Ab-MR, nucleotide sequence such as SEQ ID
Shown in NO.1.
Comprehensively consider anti insect geneCry1Ab-MRVarious characteristics and primer sensitivity, devise its cloning primer,
Nucleotide sequence such as SEQ ID NO.2 and SEQ ID NO.3；Its nucleotide sequence such as SEQ ID NO.4 and SEQ ID NO.5.
A kind of prokaryotic expression carrier comprising above-mentioned anti insect gene is had devised in conjunction with practical characteristic.
A kind of prokaryotic expression engineering bacteria for genetic transformation is constructed, which includes the anti insect gene sequence.
A kind of carrier for expression of eukaryon for Genetic Transformation in Higher Plants is further constructed, which includes the pest-resistant base
Because of sequence.
A kind of engineering bacteria for Genetic Transformation in Higher Plants is constructed, which includes the anti insect gene sequence.
By the anti insect gene, the carrier for expression of eukaryon, the application of the engineering bacteria in transgenic plants.
Preferably, the plant is monocotyledon.
Preferably, the monocotyledon is corn.
Compared with prior art, the beneficial technical effect of the present invention lies in:
1. the present invention provides a kind of anti insect gene for the first timeCry1Ab-MRThe nucleotide sequence of gene.
2. the present inventionCry1Ab-MRGene can be stablized in maize seed expresses, and overcomes foreign gene table in plant
Up to existing gene silencing phenomenon, and the insect resistace of maize leaf can be significantly improved.
3. the present inventionCry1Ab-MRGene, recombinant expression carrier and recombinant bacterium are providing plant resistance to insect with wide
Application space and market prospects.
4. of the invention by anti insect geneCry1Ab-MRAfter importing corn, the transformant of available stable heredity.In addition,
The gene can also make it have corresponding anti-insect activity with crops such as converting cotton, rice, vegetables, to reduce chemical agriculture
The usage amount of medicine has important economic value and wide application prospect to reduce environmental pollution.
Detailed description of the invention
Fig. 1 is that recombination expression vector establishment verifies electrophoretogram；
In Fig. 1,1 is PET28bNde I/HindIII double digestion, 2 be pETCry1Ab-MRNde The bis- enzymes of I/Hind III
It cuts, 3 be DNA mark SM0331, and 4 be pETCry1Ab-MR plasmid；
Fig. 2 has a try for worm and tests corn borer size comparison diagram；
In Fig. 2, A is test group, and B is control group, and C is blank group；
Fig. 3 is transgenic corn plantCry1Ab-MRGene PCR product electrophoretogram；
Fig. 4 is transgenic plantBarGene PCR product electrophoretogram；
In Fig. 3 and 4, M is DL2000 Marker；CK1 is positive control (plasmid pCAMBIA3300-Cry1Ab-MR)；CK2 is
Negative control (non-transgenic corn)；Blank is blank control (distilled water)；1~6 is transgenic corns；
Fig. 5 is corn transformation plant immunity test strip testing result figure；
In Fig. 5,1~4 is Bt-Cry1Ab/Ac test strips；1 is negative-type；2~4: transgenic line；5~8 be PAT test paper
Item；5 be negative-type；6~8 be transgenic line；
Fig. 6 is pest-resistant comparison diagram in transgenic corn plant room；
In Fig. 6,1~2 is conventional corn plant leaf；2~4 be to turnCry1Ab-MRGene corn plant leaf；
Fig. 7 is that transgenic plant and adjoining tree field insect resistace detect one of comparison diagram；
Fig. 8 is that transgenic plant and adjoining tree field insect resistace detect the two of comparison diagram；
In Fig. 7 and 8, left figure is adjoining tree, and right figure is transgenic plant.
Specific embodiment
Illustrate a specific embodiment of the invention with reference to the accompanying drawings and examples, but following embodiment is used only in detail
It describes the bright present invention in detail, does not limit the scope of the invention in any way.
Related instrument and equipment is routine instrument device unless otherwise instructed in the examples below；It is related
Bacterial strain or carrier are commercially available conventional bacterial strain or carrier unless otherwise instructed；Related test method, unless otherwise instructed,
It is conventional method.
Embodiment one: transformation anti insect geneCry1AbGene
Inventor is based on long-term a large amount of experimental study and practical experience, originalCry1Ab(GenBank Serial No.
AY847289.1 significantly Curve guide impeller) has been carried out on the basis of anti insect gene, is mainly included that codon is transformed, is confirmed and exclude
Inverted repeat sequence present in the AT enrichment region such as many places ATTTA, AATGAA and gene order in original anti insect gene DNA sequence dna
Column, remove part commonly use restriction enzyme enzyme recognition site sequence (XbaI), reduce indefinite eukaryotic DNA sequence introne
Sequence and may cause the sequence that the genetic transcription terminates in advance or causes mRNA unstable, and add termination codon at 3 ' ends
Sub- TAG, it is final to obtain the anti insect gene that codon optimization is devised according to monocotyledon coding characteristicCry1Ab-MR, core
Nucleotide sequence is as shown in SEQ ID NO.1.
Reforming composite thuringiensis insecticidal proteinsCry1Ab-MRGene, and it is originalCry1AbThe sequence of gene
It compares, has the following characteristics that
(1) AT enrichment region and the base such as many places ATTTA, AATGAA in improved anti insect gene sequence elimination original DNA sequence
Because of inverted repeats present in sequence.
(2) after transformationCry1Ab-MRIn 1848 bases of gene, A base contents are 19.64 %, and T base contents are
16.40%, C base contents are that 34.74%, G base contents are 29.22%, so that G+C content is made to be increased to 63.96% by 36.26%,
G+C content increases 27.70% on the basis of the original；
(3) homologous comparison analysis is carried out to transformation front and back anti insect gene by DNAMAN software Multiple Alignment to find
The new Bt gene of transformationCry1Ab-MRGene nucleotide series and original Bt gene homology are only 65.45%.
Embodiment two:Cry1Ab-MRGene expression in coli strain BL21 (DE3)
1. the building of prokaryotic expression carrier
(1) it using pUC carrier as frame, is synthesized and is contained by Sangon Biotech (Shanghai) Co., Ltd.Cry1Ab-MRGene
Recombinant vector is named as pUC-Cry1Ab-MR；
(2) cloning primer is designed:
Upstream primer F1:5 '-CATATGGACAACAACCCGAACATC-3 '；
Downstream primer R1:5 '-AAGCTTCTAGTACTCAGCCTCGAACGT-3 '；
Draw addition in upstreamNde I endonuclease recognized site sequence C ATATG, downstream primer additionHind The identification of III restriction endonuclease
Site sequence AAGCTT；
(3) using pUC-Cry1Ab-MR plasmid as template, above-mentioned primer amplification is utilizedCry1Ab-MRGene order；
(4) acquired PCR product is usedNdeI HeHindIII two kinds of restriction enzymes carry out double digestion, after the completion of digestion, adopt
After gel reclaims kit recovery purifying digestionCry1Ab-MRGenetic fragment；
(5) it usesNdeI HeHindIII double digestion prokaryotic expression carrier pET28b, gel reclaims kit recovery purifying 5.3kb piece
Section；
(6) after purifying digestionCry1Ab-MRGenetic fragment is attached with recovery purifying 5.3kb carrier segments reacts, building
Prokaryotic expression recombinant plasmid, is named as pET-Cry1Ab-MR；
As shown in Figure 1, passing throughNdeI HeHindThe digestion verification of III two kinds of restriction enzymes shows that vector construction is correct.
2. the building of recombination bacillus coli
Recombinant plasmid pET-Cry1Ab-MR is converted into Escherichia coliE.coliBL21 (DE3), filters out positive transformant.It extracts
It after plasmid enzyme restriction verifying, selects positive transformant and is seeded in resistance culture base, 37 DEG C are incubated overnight, and are turned with 2% inoculum concentration
It connects, culture to OD600Value about saves backup for 0.5~0.6,4 DEG C.
3. detecting insect resistace in recombinant bacterium expression product room
(1) test process is divided into three groups:
By recombination bacillus coli after IPTG is induced, ultrasonic disruption is carried out, the supernatant of collection is as test group；Under the same terms
Cultivate pET28b containing empty carrier E.coliBL21 (DE3) carries out ultrasonic disruption, and the supernatant of collection is as a control group；With clear
Water is as blank group.
(2) three groups of liquid of equivalent is taken to be added in the corn borer feed of configuration as test feed, raising corn borer into
The examination of row worm.
Specific steps are as follows:
Each test tube is put into a feed, and is respectively connected to 10 corn borer larvaes；Each processing respectively connects 10 test tubes；It is put into temperature
26~28 DEG C are spent, is cultivated 8 days in the environment of relative humidity 70% or so, detection average mortality and single weight of worm living.
Concrete outcome is shown in Table 1:
1 worm of table tries test result
。
1 statistical result of table shows:Cry1Ab-MRThe Bt insecticidal proteins of gene coding have very strong insecticidal effect, corn
The death rate of snout moth's larva is up to 91.67%, and has apparent inhibiting effect to the growth of corn borer, and worm living is single only to only have 0.1426mg again.
After culture 8 days, picking is hidden in the corn borer in man-made feeds, and it is big that collection is put into observation corn borer in culture dish
It is small.
It is as shown in Figure 2: can clearly to find the corn borer insect size of blank group more than 1 centimetre；Corn in control group
1 centimetre of snout moth's larva size or so；And the corn borer insect size of test group hatches almost indifference with rigid.
From the above prokaryotic expression insect bioassay:Cry1Ab-MRThe Bt insecticidal proteins of gene coding have very strong kill
Worm effect, explanationCry1Ab-MRGenetic modification can give expression to the toxic protein with strong biological activity.
Embodiment three: buildingCry1Ab-MRThe recombinant expression carrier of gene and recombinant expression bacterium
Specific step is as follows:
(1) it using carrier T as frame, is synthesized and is contained by Sangon Biotech (Shanghai) Co., Ltd.Cry1Ab-MRThe weight of gene
Group carrier, is named as T-Cry1Ab-MR；
(2) it using T-Cry1Ab-MR as template, is cloned using amplimerCry1Ab-MRGene；
Upstream primer containsXbaI restriction enzyme site, primer sequence are as follows:
Cry1Ab-MR-F:TCTAGAATGGACAACAACCCGAACATC；
Downstream primer containsSacI restriction enzyme site, primer sequence are as follows:
Cry1Ab-MR-R:GAGCTCCTAGTACTCAGCCTCGAACG；
(3) it usesXbaI andSacI double digestion clone'sCry1Ab-MRProduct, recycling genetic fragment；
(4) it is connected into and has usedXbaI 、SacOn the genetic transformation high-efficiency plant expression vector pCAMBIA3300 of I double digestion processing,
?Cry1Ab-MRThe recombinant expression carrier of gene, recombinant plasmid are named as pCAMBIA3300- Cry1Ab-MR；
(5) recombinant plasmid pCAMBIA3300-Cry1Ab-MR is converted into Agrobacterium EHA105, screens positive strain, obtainsCry1Ab-MRThe recombinant expression bacterium of gene, low-temperature preservation are used for follow-up test.
Example IV: recombinational agrobacterium mediated transformation maize immature embryos and callus
1. removing maize immature embryos
(1) bracteal leaf of corn is removed；
(2) fruit ear top about 1cm or so is cut off, then fruit ear is put with tweezers as handle from top insertion fruit ear with tweezers
Enter in the beaker containing thimerosal, according to actual needs, 4~6 fruit ears can be put in the same beaker；
(3) in beaker plus the thimerosal of about 700mL (50% bleaching agent or 5.25% sodium hypochlorite, and a drop Tween is added
20) fruit ear is impregnated, 20min is sterilized；During disinfection, rotation fruit ear frequently pats beaker gently simultaneously to drive away seed surface
Bubble, to reach optimal Disinfection Effect；
(4) it after sterilizing, takes out fruit ear and is put into the beaker for filling with aqua sterilisa, washed in water 3 times, be ready for stripping embryo；
(5) one end for sterilizing fruit ear is placed on a big culture dish, the top (1.5 of seed is reamed with big scalpel
~1.8mm), during this process, tool used is frequently sterilized, such as: knife blade, culture dish, stripping embryo knife；With stripping embryo knife
Point of a knife be inserted between embryo and endosperm, then gently pull out rataria upwards, gently hold up rataria with small operation point of a knife, it is ensured that children
Embryo is close to the plumular axis face of rataria to be placed with the N6E culture medium of filter paper, the density of embryo is about 2 × 2cm not by any damage
(30/ware)；
(6) ParafilmTM culture dish, 28 DEG C dark culture 2~3 days.
2. recombinational agrobacterium infects
(1) recombinational agrobacterium that Example three constructs is in YEP(50 mg/L and Str100 mg/L antibiotic containing Kan) culture
Activation culture on base；
(2) streak inoculation is cultivated 3 days in YEP culture medium (the 50 mg/L antibiotic containing Kan 50 mg/L and Str) at 19 DEG C；
(3) picking recombinational agrobacterium is put into the 50 mL centrifuge tubes containing 5 mL dip dyeing culture medium, while adding 100 uM AS
(inf+AS), 2~4 h of bacterium is shaken in 75 rpm of room temperature (25 DEG C) revolving speed；
(4) rataria of removing is put into the centrifuge tube containing inf+AS fluid nutrient medium (2 mL), about 20~100 childrens of every pipe
Embryo is washed 2 times with such culture medium, 1~1.5 mL OD is then added550=0.3~0.4 recombinational agrobacterium, gently overturns
Centrifuge tube 20 times, then uprightly it is placed on 5 min in camera bellows, it is ensured that rataria is all immersed in Agrobacterium liquid, whole process
Vortex is avoided to vibrate.
3. co-culturing
After infecting, the rataria disseminated is transferred to co-culture medium, so that the plumular axis of rataria is contacted media surface, uses simultaneously
Aseptic filter paper blots, and drives away the extra Agrobacterium of media surface；With ParafilmTM culture dish, dark culture at 20 °C
3 days.
4. tranquillization
After co-culturing 3 days, rataria is transferred to above tranquillization culture medium, while with ParafilmTM culture dish, being placed on 28 DEG C of items
Dark culture 7 days under part.
5. selection
After 7 days, all ratarias are transferred to above Selective agar medium (35/ware), are cultivated two weeks, Selective agar medium contains
Double third ammonia phosphorus of 1.5 mg/L carry out subculture again after two weeks, and the concentration of double third ammonia phosphorus can rise to 3 mg/L, and dip dyeing is about
5 weeks or so, the cell containing transformant may have grown into visible II type callus.
6. the regeneration of transgenic plant
In illumination cultivation room, takes callus 3 weeks long on regeneration culture medium I, then germinate on regeneration culture medium II；
When regenerated transgenic seedling grows 3~4 leaves, it is transferred into greenhouse, and checked, positive plant retains.To its life
When the long extremely spinning loose powder phase, pollinate to it.
Embodiment five: detectionCry1Ab-MRExpression of the gene in plant
1. PCR is detected
When the transgenic plant that example four to be performed is cultivated grew into for 5~6 leaf phase, CTAB method extracts the leaves genomic DNA of plant,
Design primer carries out PCR amplificationCry1Ab-MRGlufosinate-resistant gene on gene and plasmid pCAMBIA3300barGene.
FoundationCry1Ab-MRGene andbarThe PCR detection primer sequence of gene internal sequence design is as follows:
Cry1Ab-MR-F ': CGAGGGAGATTTACACGAAC；
Cry1Ab-MR-R ': GGTTGCTGCTGGTGCCGTAC；
Bar-F:ATGAGCCCAGAACGACGCC；
Bar-R:TTAGATCTCGGTGACGGGC.
The reaction system of PCR are as follows: the DNA template of 2ul, 2uL10 × PCR Buffer buffer, the dNTP of 2ul
(10mM each), 1ul upstream primer (10mM), 1uL downstream primer (10mM), 0.3uL Tap enzyme, sterile water supply 20 ul.
The response procedures of PCR are as shown in table 2:
2 PCR response procedures of table
。
Testing result is as shown in Figure 3 and Figure 4:
Corn gene plant PCR is positive findings, amplifies the piece of 552 bp of segment and size of size 396bp respectively
Section.Negative control and blank control do not show corresponding band, it was demonstrated that the target fragment that PCR amplification goes out is purpose gene really
Internal sequence, it was demonstrated that foreign geneCry1Ab-MRGene andbarGene has been integrated into Maize genome.
2. test strips detect
Transgenic line is tested using Bt-Cry1Ab/Ac, PAT immunity test strip, to analyze anti insect geneCry1Ab- MRWith selected marker albumen PAT encoding genebarGenetic stability and target protein expression.
Pest-resistant test strips use Cry1Ab/Ac test strips (the article No. STX 06200/ purchased from the production of U.S. Agdia company
0050) Cry1Ab protein expression, the same product description of concrete operations are detected.
Bar test strips use the pat/bar test strips (article No. purchased from the production of department, Agdia company, the U.S. U.S. E
AS013LS013 marker gene) is detectedbarExpression of gene protein, the same product description of concrete operations.
Testing result is as shown in Figure 5: it is detected with insect resistance protein Cry1Ab-t and selected marker albumen PAT test strips,
There is red detection line quickly in the lower section of nature controlling line in corn transformation body plant, and compares nontransgenic plants accordingly only
There is a red nature controlling line.
As shown in Figure 5:Cry1Ab-MRWithbarThe destination protein of gene has expression in corn transformation body, and compares non-turn
Gene plant is not expressed.Prove foreign geneCry1Ab-MRIt has been integrated into Maize genome, is avoided that gene " silencing " phenomenon
Appearance.Transgenic plant material insect resistance protein has high level expression, and apparent altitude stability, right in transgenic plant material
It is not expressed according to nontransgenic plants.
Embodiment five: the insect resistace in identification transgenic corn plant
1. turnCry1Ab-MRThe identification of anti insect gene Maize at Seedling Stage blade Ostrinia furnacalis resistance indoor biometrics
It takes and turns plant growth of seedling to 5~8 leaf phase plant aerial parts and take back interior, take the tender heart of children undeployed
Leaf is cut into 2~3 cm sizes with disinfection scissors, is placed in 24 porocyte culture plates, and every hole connects 3 newly hatched larvaes.With common beautiful
Rice plant leaf is control group, is turnedCry1Ab-MRGene plant blade is test group, observes transgenic corns after culture 1 week
Insect resistant effect.
It is as shown in Figure 6:
The conventional corn blade of control group is almost suffered all by corn borer, and resistance rank is high sense；And turnCry1Ab-MRGene plant
Blade resistance effect is good, and plant shows the resistance of height.
2. turnCry1Ab-MRAnti insect gene maize leaf Artificial Inoculation of Anoplophora glabripennis field corn snout moth's larva Resistance Identification
After corn growth one month, generally 6~8 leaf phases, plant height starts Artificial Inoculation of Anoplophora glabripennis corn borer at 30 centimeters or so.Often
A centrifuge tube puts 2~3 pieces of pieces of an egg, the centrifuge tube for installing worm's ovum is put into 28 DEG C of incubator and is cultivated；When inoculation, open just
Centrifuge tube is inserted into non-transgenic and transgenic corns lobus cardiacus by the centrifuge tube for hatching larva.It adjusts within 20~25 days after connecing worm
Look into food leaf level.The record method for eating leaf level is carried out referring to 720.1 ~ 720.3-2003 of Ministry of Agriculture's professional standard NY/T.
It is as shown in Figure 7 and Figure 8:
Non-transgenic control material blade worm food is serious, and transgenic line insect resistant effect is significant.
Insect resistace comparison further is carried out to corn gene material, analyze transgenosis and its corresponding control food leaf level and
Insect pest rank, corn gene material insect resistace carry out evaluation of resistance.
Corn lobus cardiacus is as shown in table 3 by the grade scale of the corn borer extent of injury:
Grade scale of the 3 corn lobus cardiacus of table by the corn borer extent of injury
。
Corn is as shown in table 4 to the Evaluation standard of resistance of corn borer:
Evaluation standard of resistance of 4 corn of table to corn borer
。
The results are shown in Table 5:
The identification of 5 leaf efficacy of table
5% level goes up significant difference between different lowercases in table, and numerical value is Mean ± SE in table.
It is as shown in table 5: to there is more quantity to be greater than the worm channel of 2mm, edible grade on non-transgenic material partial blade
Reach 9 grades, food leaf level is significantly higher than corresponding transgenic line in 0.05 level, and it is horizontal to reach high sense；And transgenosis
Material does not have have a small amount of needle prick shape (≤1 mm) worm channel on worm channel or only individual young leaves, and insect pest rank reaches highly resistance level, field
Pest-resistant sex expression is stablized.
It is as follows to analyze result: control material worm food is serious, and insect pest rank reaches 9 grades, and the pest-resistant effect of corn gene material
Fruit is obvious.
The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art
Member is it is understood that without departing from the purpose of the present invention, can also carry out each design parameter in above-described embodiment
Change, forms multiple specific embodiments, is common variation range of the invention, is no longer described in detail one by one herein.
SEQUENCE LISTING
<110>Henan Academy of Agricultural Sciences
<120>gene C ry1Ab-MR, its expression vector and its application of Bt insecticidal proteins are encoded
<130> 2019
<160> 9
<170> PatentIn version 3.2
<210> 1
<211> 1848
<212> DNA
<213>artificial synthesized
<400> 1
atggacaaca acccgaacat caacgagtgc atcccctaca actgcctgag caaccccgag 60
gtcgaggtcc tcggaggcga gcggatcgag accggctaca cccccatcga catcagcctg 120
tcgctcacgc agttcctcct gtccgaattc gtgcccggcg ccggcttcgt gctgggcctg 180
gtcgacatca tctgggggat cttcgggccg agccagtggg acgccttcct ggtgcagatc 240
gagcaactca tcaaccagcg gatcgaggaa ttcgcccgca accaggccat cagccgcctg 300
gaggggctct ccaacttgta ccagatctac gccgagagct tccgcgagtg ggaggccgac 360
ccgacgaatc cggcgttgag ggaagagatg cgcatccagt tcaacgacat gaacagcgcc 420
ctcacgacgg cgatcccgct cttcgcggtc cagaattacc aggtgcccct gctgagcgtg 480
tatgtccagg cggcgaacct ccatttgtcg gtgctgcgcg acgtcagcgt gttcggccag 540
cgctgggggt tcgacgcggc gacgatcaac agccgctaca acgacctgac ccgcctgatc 600
gggaactaca cggatcacgc ggtccggtgg tacaacaccg gcctggagcg cgtgtggggt 660
ccggactcca gggactggat ccgctacaac cagttccgcc gcgagctgac cctgaccgtg 720
ctcgatatcg tcagcttgtt ccctaactac gacagccgca cctaccccat ccgcaccgtg 780
tcgcagctca cgagggagat ttacacgaac cccgtgctgg agaacttcga cggcagcttc 840
cgggggtccg cgcaggggat cgaggggtcg atccgcagcc cccacctgat ggacatcctg 900
aactcgatca cgatctacac ggacgcgcac cgcggcgagt actactggag cggccaccag 960
atcatggcgt cgccggtggg cttctcgggc cccgagttca ccttccccct gtacggcacc 1020
atggggaacg cggccccgca gcagcggatc gtggcacagc tgggccaggg agtgtaccgc 1080
acgctcagca gcacgctcta ccgccgcccg ttcaacatcg gcatcaacaa ccagcagctg 1140
tcggtcctcg atgggacgga gttcgcgtac ggcaccagca gcaacctgcc cagcgccgtg 1200
taccggaagt cagggacggt cgactcgctc gacgagatcc cccctcagaa caacaacgtg 1260
ccgccgcggc aggggttctc gcaccggctc agccacgtga gcatgttccg cagtggcttc 1320
tcgaactcgt cggtctcgat catccgcgcg cctatgttca gctggattca ccgcagtgcc 1380
gaattcaaca acatcattcc gtcgtcgcag atcacccaga tccccctgac caagagcacc 1440
aacctcgggt cggggacgtc ggtcgtcaag ggccccggct tcaccggcgg cgacatcctg 1500
cggcggacga gcccggggca gatctcgaca ctgcgcgtga acatcaccgc ccccctgagc 1560
cagcgctacc gggtgcgaat ccggtacgcg agcaccacca acctgcagtt ccacaccagc 1620
atcgacggtc ggccgatcaa ccagggaaac ttcagcgcca ccatgagcag cggcagcaac 1680
ctccagtcgg gttcgttccg gacggtaggc ttcaccaccc ccttcaactt cagcaacggc 1740
tcgtcggtct tcacgctctc ggcgcacgtc ttcaacagcg gcaacgaggt gtacatcgac 1800
aggatcgagt tcgtcccggc ggaggtcacg ttcgaggctg agtactag 1848
<210> 2
<211> 24
<212> DNA
<213>artificial synthesized
<400> 2
catatggaca acaacccgaa catc 24
<210> 3
<211> 27
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<213>artificial synthesized
<400> 3
aagcttctag tactcagcct cgaacgt 27
<210> 4
<211> 27
<212> DNA
<213>artificial synthesized
<400> 4
tctagaatgg acaacaaccc gaacatc 27
<210> 5
<211> 26
<212> DNA
<213>artificial synthesized
<400> 5
gagctcctag tactcagcct cgaacg 26
<210> 6
<211> 20
<212> DNA
<213>artificial synthesized
<400> 6
cgagggagat ttacacgaac 20
<210> 7
<211> 20
<212> DNA
<213>artificial synthesized
<400> 7
ggttgctgct ggtgccgtac 20
<210> 8
<211> 19
<212> DNA
<213>artificial synthesized
<400> 8
atgagcccag aacgacgcc 19
<210> 9
<211> 19
<212> DNA
<213>artificial synthesized
<400> 9
ttagatctcg gtgacgggc 19
Claims (9)
1. a kind of anti insect geneCry1Ab-MR, nucleotide sequence is as shown in SEQ ID NO.1.
2. anti insect gene described in a kind of clone's claim 1Cry1Ab-MRPrimer, which is characterized in that its nucleotide sequence is such as
SEQ ID NO.2 and SEQ ID NO.3；Its nucleotide sequence such as SEQ ID NO.4 and SEQ ID NO.5.
3. a kind of prokaryotic expression carrier for genetic transformation, which is characterized in that the carrier includes described in claim 1 pest-resistant
GeneCry1Ab-MR。
4. a kind of prokaryotic expression engineering bacteria for genetic transformation, which is characterized in that the engineering bacteria includes described in claim 1
Anti insect geneCry1Ab-MR。
5. a kind of carrier for expression of eukaryon for Genetic Transformation in Higher Plants, which is characterized in that the carrier includes described in claim 1
Anti insect geneCry1Ab-MR。
6. a kind of engineering bacteria for Genetic Transformation in Higher Plants, which is characterized in that the engineering bacteria includes described in claim 1 pest-resistant
Gene.
7. carrier for expression of eukaryon described in anti insect gene described in claim 1, claim 5, engineering as claimed in claim 6
The application of any one of bacterium claim in transgenic plants.
8. application according to claim 7, which is characterized in that the plant is monocotyledon.
9. application according to claim 8, which is characterized in that the monocotyledon is corn.
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| CN111041036A (en) * | 2019-12-16 | 2020-04-21 | 河南省农业科学院 | Encoding insecticidal protein insect-resistant fusion gene mCryAb-VIP3A, expression vector and application thereof |
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